Varespladib Inhibits the Phospholipase A2 and Coagulopathic Activities of Venom Components from Hemotoxic Snakes

Phospholipase A2 (PLA2) enzymes are important toxins found in many snake venoms, and they can exhibit a variety of toxic activities including causing hemolysis and/or anticoagulation. In this study, the inhibiting effects of the small molecule PLA2 inhibitor varespladib on snake venom PLA2s was investigated by nanofractionation analytics, which combined chromatography, mass spectrometry (MS), and bioassays. The venoms of the medically important snake species Bothrops asper, Calloselasma rhodostoma, Deinagkistrodon acutus, Daboia russelii, Echis carinatus, Echis ocellatus, and Oxyuranus scutellatus were separated by liquid chromatography (LC) followed by nanofractionation and interrogation of the fractions by a coagulation assay and a PLA2 assay. Next, we assessed the ability of varespladib to inhibit the activity of enzymatic PLA2s and the coagulopathic toxicities induced by fractionated snake venom toxins, and identified these bioactive venom toxins and those inhibited by varespladib by using parallel recorded LC-MS data and proteomics analysis. We demonstrated here that varespladib was not only capable of inhibiting the PLA2 activities of hemotoxic snake venoms, but can also effectively neutralize the coagulopathic toxicities (most profoundly anticoagulation) induced by venom toxins. While varespladib effectively inhibited PLA2 toxins responsible for anticoagulant effects, we also found some evidence that this inhibitory molecule can partially abrogate procoagulant venom effects caused by different toxin families. These findings further emphasize the potential clinical utility of varespladib in mitigating the toxic effects of certain snakebites.

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Antivenom Neutralization of Coagulopathic Snake Venom Toxins Assessed by Bioactivity Profiling Using Nanofractionation Analytics

Toxins ◽

10.3390/toxins12010053 ◽

2020 ◽

Vol 12 (1) ◽

pp. 53 ◽

Cited By ~ 6

Author(s):

Chunfang Xie ◽

Julien Slagboom ◽

Laura-Oana Albulescu ◽

Ben Bruyneel ◽

Kristina B. M. Still ◽

...

Keyword(s):

Snake Venom ◽

Serine Proteases ◽

Clinical Symptoms ◽

Neglected Tropical Diseases ◽

Anticoagulant Activity ◽

Proteomics Data ◽

Neutralization Capacity ◽

Venom Toxins ◽

Coagulation Assay ◽

Deinagkistrodon Acutus

Venomous snakebite is one of the world’s most lethal neglected tropical diseases. Animal-derived antivenoms are the only standardized specific therapies currently available for treating snakebite envenoming, but due to venom variation, often this treatment is not effective in counteracting all clinical symptoms caused by the multitude of injected toxins. In this study, the coagulopathic toxicities of venoms from the medically relevant snake species Bothrops asper, Calloselasma rhodostoma, Deinagkistrodon acutus, Daboia russelii, Echis carinatus and Echis ocellatus were assessed. The venoms were separated by liquid chromatography (LC) followed by nanofractionation and parallel mass spectrometry (MS). A recently developed high-throughput coagulation assay was employed to assess both the pro- and anticoagulant activity of separated venom toxins. The neutralization capacity of antivenoms on separated venom components was assessed and the coagulopathic venom peptides and enzymes that were either neutralized or remained active in the presence of antivenom were identified by correlating bioassay results with the MS data and with off-line generated proteomics data. The results showed that most snake venoms analyzed contained both procoagulants and anticoagulants. Most anticoagulants were identified as phospholipases A2s (PLA2s) and most procoagulants correlated with snake venom metalloproteinases (SVMPs) and serine proteases (SVSPs). This information can be used to better understand antivenom neutralization and can aid in the development of next-generation antivenom treatments.

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Neutralizing Effects of Small Molecule Inhibitors and Metal Chelators on Coagulopathic Viperinae Snake Venom Toxins

Biomedicines ◽

10.3390/biomedicines8090297 ◽

2020 ◽

Vol 8 (9) ◽

pp. 297 ◽

Cited By ~ 2

Author(s):

Chunfang Xie ◽

Laura-Oana Albulescu ◽

Mátyás A. Bittenbinder ◽

Govert W. Somsen ◽

Freek J. Vonk ◽

...

Keyword(s):

Phospholipase A2 ◽

Small Molecule ◽

Snake Venom ◽

Small Molecule Inhibitors ◽

Drug Repurposing ◽

Proteomics Data ◽

Snake Venoms ◽

Analytical Technique ◽

Venom Toxins ◽

Snake Venom Metalloproteinase

Animal-derived antivenoms are the only specific therapies currently available for the treatment of snake envenoming, but these products have a number of limitations associated with their efficacy, safety and affordability for use in tropical snakebite victims. Small molecule drugs and drug candidates are regarded as promising alternatives for filling the critical therapeutic gap between snake envenoming and effective treatment. In this study, by using an advanced analytical technique that combines chromatography, mass spectrometry and bioassaying, we investigated the effect of several small molecule inhibitors that target phospholipase A2 (varespladib) and snake venom metalloproteinase (marimastat, dimercaprol and DMPS) toxin families on inhibiting the activities of coagulopathic toxins found in Viperinae snake venoms. The venoms of Echis carinatus, Echis ocellatus, Daboia russelii and Bitis arietans, which are known for their potent haemotoxicities, were fractionated in high resolution onto 384-well plates using liquid chromatography followed by coagulopathic bioassaying of the obtained fractions. Bioassay activities were correlated to parallel recorded mass spectrometric and proteomics data to assign the venom toxins responsible for coagulopathic activity and assess which of these toxins could be neutralized by the inhibitors under investigation. Our results showed that the phospholipase A2-inhibitor varespladib neutralized the vast majority of anticoagulation activities found across all of the tested snake venoms. Of the snake venom metalloproteinase inhibitors, marimastat demonstrated impressive neutralization of the procoagulation activities detected in all of the tested venoms, whereas dimercaprol and DMPS could only partially neutralize these activities at the doses tested. Our results provide additional support for the concept that combinations of small molecules, particularly the combination of varespladib with marimastat, serve as a drug-repurposing opportunity to develop new broad-spectrum inhibitor-based therapies for snakebite envenoming.

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High throughput screening and identification of coagulopathic snake venom proteins and peptides using nanofractionation and proteomics approaches

10.1101/780155 ◽

2019 ◽

Cited By ~ 2

Author(s):

Julien Slagboom ◽

Marija Mladić ◽

Chunfang Xie ◽

Freek Vonk ◽

Govert W. Somsen ◽

...

Keyword(s):

Mass Spectrometry ◽

Blood Coagulation ◽

Snake Venom ◽

High Throughput ◽

High Throughput Screening ◽

Neglected Tropical Disease ◽

Snake Venoms ◽

Plasma Coagulation ◽

Venom Toxins ◽

Coagulation Assay

AbstractSnakebite is a neglected tropical disease that results in a variety of systemic and local pathologies in envenomed victims and is responsible for around 138,000 deaths every year. Many snake venoms cause severe coagulopathy that makes victims vulnerable to suffering life-threating haemorrhage. The mechanisms of action of coagulopathic snake venom toxins are diverse and can result in both anticoagulant and procoagulant effects. However, because snake venoms consist of a mixture of numerous protein and peptide components, high throughput characterizations of specific target bioactives is challenging. In this study, we applied a combination of analytical and pharmacological methods to identify snake venom toxins from a wide diversity of snake species that perturb coagulation. To do so, we used a high-throughput screening approach consisting of a miniaturised plasma coagulation assay in combination with a venom nanofractionation approach. Twenty snake venoms were first separated using reversed-phase liquid chromatography, and a post-column split allowed a small fraction to be analyzed with mass spectrometry, while the larger fraction was collected and dispensed onto 384-well plates before direct analysis using a plasma coagulation assay. Our results demonstrate that many snake venoms simultaneously contain both procoagulant and anticoagulant bioactives that contribute to coagulopathy. In-depth identification analysis from seven medically-important venoms, via mass spectrometry and nanoLC-MS/MS, revealed that phospholipase A2 toxins are frequently identified in anticoagulant venom fractions, while serine protease and metalloproteinase toxins are often associated with procoagulant bioactivities. The nanofractionation and proteomics approach applied herein seems likely to be a valuable tool for the rational development of next-generation snakebite treatments by facilitating the rapid identification and fractionation of coagulopathic toxins, thereby enabling specific targeting of these toxins by new therapeutics such as monoclonal antibodies and small molecule inhibitors.Author summarySnakebite is a neglected tropical disease that results in more than 100,000 deaths every year. Haemotoxicity is one of the most common signs of systemic envenoming observed after snakebite, and many snake venoms cause severe impairment of the blood coagulation that makes victims vulnerable to suffering life-threating hemorrhage. In this study, we applied a combination of analytical and pharmacological methods to identify snake venom toxins from a wide diversity of snake species that interfere with blood coagulation. Twenty snake venoms were screened for their effects on the blood coagulation cascade and based on the initial results and the medical relevance of the species, seven venoms were selected for in-depth analysis of the responsible toxins using advanced identification techniques. Our findings reveal a number of anticoagulant toxins that have not yet been reported before as such. The methodology described herein not only enables the identification of both known and unknown toxins that cause impairment of the blood coagulation, but offers a throughput platform to effectively screen for inhibitory molecules relevant for the development of next generation snakebite treatments.

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Broad cytolytic specificity of myotoxin II, a lysine-49 phospholipase A2 of Bothrops asper snake venom

Toxicon ◽

10.1016/0041-0101(94)90408-1 ◽

1994 ◽

Vol 32 (11) ◽

pp. 1359-1369 ◽

Cited By ~ 69

Author(s):

Bruno Lomonte ◽

Andrej Tarkowski ◽

Lars Å. Hanson

Keyword(s):

Phospholipase A2 ◽

Snake Venom ◽

Bothrops Asper

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Immunochemical properties of the N-terminal helix of myotoxin II, a lysine-49 phospholipase A2 from Bothrops asper snake venom

Toxicon ◽

10.1016/s0041-0101(00)00227-0 ◽

2001 ◽

Vol 39 (6) ◽

pp. 879-887 ◽

Cited By ~ 16

Author(s):

Yamileth Angulo ◽

Carlos E Núñez ◽

Sergio Lizano ◽

Andreimar M Soares ◽

Bruno Lomonte

Keyword(s):

Phospholipase A2 ◽

Snake Venom ◽

Bothrops Asper

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Effect of modification of one histidine residue on the enzymatic and pharmacological properties of a toxic phospholipase A2 from Naja nigricollis snake venom and less toxic phospholipases A2 from Hemachatus haemachatus and Naja naja atra snake venoms

Toxicon ◽

10.1016/0041-0101(81)90118-5 ◽

1981 ◽

Vol 19 (1) ◽

pp. 61-71 ◽

Cited By ~ 76

Author(s):

Eleonora Condrea ◽

Jeffrey E. Fletcher ◽

Bruce E. Rapuano ◽

Chen-Chung Yang ◽

Philip Rosenberg

Keyword(s):

Phospholipase A2 ◽

Snake Venom ◽

Pharmacological Properties ◽

Histidine Residue ◽

Snake Venoms ◽

Naja Naja ◽

Phospholipases A2 ◽

Naja Atra

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A phospholipase A2 from Bothrops asper snake venom activates neutrophils in culture: Expression of cyclooxygenase-2 and PGE2 biosynthesis

Toxicon ◽

10.1016/j.toxicon.2010.12.004 ◽

2011 ◽

Vol 57 (2) ◽

pp. 288-296 ◽

Cited By ~ 10

Author(s):

Vanessa Moreira ◽

José María Gutiérrez ◽

Rafaela Bacci Amaral ◽

Bruno Lomonte ◽

Eduardo Purgatto ◽

...

Keyword(s):

Phospholipase A2 ◽

Snake Venom ◽

Cyclooxygenase 2 ◽

Bothrops Asper

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Anticoagulant Activity of Naja nigricollis Venom Is Mediated by Phospholipase A2 Toxins and Inhibited by Varespladib

Toxins ◽

10.3390/toxins13050302 ◽

2021 ◽

Vol 13 (5) ◽

pp. 302

Author(s):

Taline D. Kazandjian ◽

Arif Arrahman ◽

Kristina B. M. Still ◽

Govert W. Somsen ◽

Freek J. Vonk ◽

...

Keyword(s):

Phospholipase A2 ◽

Anticoagulant Activity ◽

West African ◽

Snake Venoms ◽

The West ◽

Coagulation Assays ◽

Venom Toxins ◽

African Black ◽

Snake Venom Metalloproteinase ◽

Species Specific

Bites from elapid snakes typically result in neurotoxic symptoms in snakebite victims. Neurotoxins are, therefore, often the focus of research relating to understanding the pathogenesis of elapid bites. However, recent evidence suggests that some elapid snake venoms contain anticoagulant toxins which may help neurotoxic components spread more rapidly. This study examines the effects of venom from the West African black-necked spitting cobra (Naja nigricollis) on blood coagulation and identifies potential coagulopathic toxins. An integrated RPLC-MS methodology, coupled with nanofractionation, was first used to separate venom components, followed by MS, proteomics and coagulopathic bioassays. Coagulation assays were performed on both crude and nanofractionated N. nigricollis venom toxins as well as PLA2s and 3FTx purified from the venom. Assays were then repeated with the addition of either the phospholipase A2 inhibitor varespladib or the snake venom metalloproteinase inhibitor marimastat to assess whether either toxin inhibitor is capable of neutralizing coagulopathic venom activity. Subsequent proteomic analysis was performed on nanofractionated bioactive venom toxins using tryptic digestion followed by nanoLC-MS/MS measurements, which were then identified using Swiss-Prot and species-specific database searches. Varespladib, but not marimastat, was found to significantly reduce the anticoagulant activity of N. nigricollis venom and MS and proteomics analyses confirmed that the anticoagulant venom components mostly consisted of PLA2 proteins. We, therefore, conclude that PLA2s are the most likely candidates responsible for anticoagulant effects stimulated by N. nigricollis venom.

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